This is a proposal to study regulated vs. constitutive protein secretion in two established secretory cell lines, the rat PC12 and mouse AtT20 neuroendocrine cells. The biosynthetic-secretory pathway controls the synthesis and delivery of proteins and other molecules to lysosomes, the plasma membrane, and the cell exterior. The accurate trafficking of proteins through this pathway depends upon sorting and organelle-based transport processes that direct and deliver proteins to their appropriate destinations. Neuroendocrine cells, as a secretory cell type, have two secretory pathways: regulated and constitutive. The proposed studies will examine sorting and transport of a regulated secretory protein, tissue plasminogen activator (tPA) in the neuroendocrine cells using fluorescent hybrid proteins and time-lapse, multi-color, wide-field fluorescence microscopy. In specific aim 1, signals on tPA responsible for it's sorting will be dual-color fluorescence microscopy, deletion mutants, and a fluorescent hybrid protein, tPA/GFP, consisting of tPA and enhanced green fluorescent protein. In specific aim 2, the putative roles that cytoskeletal constituents play in dictating transport and exocytosis within the regulated pathway will be probed by labeling tPA with enhanced cyan fluorescent protein and actin or tubulin with enhanced yellow fluorescent protein and visualizing the resulting double-labeled samples using dual-color, time-lapse fluorescent microscopy. In specific aim 3, the biogenesis from the trans-Golgi network and subsequent dynamics of the organelles that transport tPA/GFP through the regulated pathway will be characterized using time-lapse fluorescence microscopy. The results obtained from these studies will provide insight into sorting and transport processes within neuroendocrine cells and may help to elucidate how mistakes in these processes can lead to mistargeting of proteins and associated cellular disorder or disease.